The present invention relates to protease proteins, nucleic acids encoding such proteins, and methods of treatment using such proteins and polynucleotides.
Complex multicellular organisms employ numerous mechanisms to protect and maintain the integrity of cellular components from which they are comprised. In the event of questionable or irreparable cell damage, one seemingly drastic measure available to a compromised cell is to commit suicide and to discretely remove itself for the altruistic benefit of the organism as a whole. Cell suicide is triggered in response to pathogenic invasion (such as by a virus) to halt the spread to neighboring cells, but it also occurs under nonthreatening conditions to replace redundant or unnecessary cells, as occurs in tissue morphogenesis and remodeling, and in the normal turnover of cells that occurs when they have exhausted their overall usefulness within the organism. Nicholson, Nature Biotechnology 14:297 (1996).
The pathological manifestation of cell suicide, known as xe2x80x9capoptosisxe2x80x9d, occurs as a result of a highly systematic and deliberate cell death pathway. These types of cell deaths have been appropriately termed xe2x80x9cphysiological cell deathxe2x80x9d, where in addition to this suicide pathway, death occurs in specific cells at a predetermined time. Apoptotic death is a highly ordered process that is characterized by nuclear changes such as chromatin condensation, fragmentation and margination as well as internucleosomal DNA cleavage (usually resulting in the hallmark DNA laddering), and by ultrastructural changes including cytoskeletal disruption, cell shrinkage and membrane blebbing which then leads to fragmentation of the dying cell into numerous membrane-bound apoptotic bodies that are subsequently engulfed by neighboring cells or professional macrophages in the final resolution of the suicide process. Apoptotic suicide has many advantages over other forms of cell death, owing principally to the membrane integrity that is maintained throughout the entire process. Necrotic cells, for example, leak their constituents into the surrounding extracellular space usually resulting in an inflammatory response. Nicholson, supra.
The recent explosion in interest in apoptosis is warranted given the substantial evidence that inappropriate apoptosis may contribute to the pathology of several human diseases (including without limitation neurological diseases, such as Alzheimer""s disease, amyotrophic lateral sclerosis, spinal muscular atrophy, neurological stroke damage, Parkinson""s disease and Huntington""s disease; immune system disorders, such as autoimmune syndromes, AIDS and type I diabetes; cardiovascular conditions, such as ischemic cardiac damage; proliferative conditions, such as solid tumors, lymphomas (such as follicular lymphoma) and leukemias; and others, such as pathogenic (including viral) infections, alopecia and aging. These can be divided into disorders of excessive apoptosis (such as neurodegenerative disease or ischemic damage) and those where insufficient apoptosis occurs (such as autoimmune syndromes, cancers and sustained pathogenic infections). Nicholson, supra.
In contrast to the above mentioned disorders, insufficient apoptosis is also associated with several human diseases. For example, many cancers are now believed to be the consequence of failed apoptotic cell death instead of enhanced cell growth as was originally thought. Two gene defects that are highly associated with proliferative disorders (p53 and Bcl-2) are now known to be regulators of the apoptotic process. Another example of flawed apoptosis is in autoimmune disorders where the failure to remove autoreactive lymphocytes that arise during development, or subsequent to an immune response, occurs. One form of autoimmune disease, lupus erythematosus, may involve failure to complete apoptosis execution since most lupus autoantibodies recognize cryptic epitopes within a distinct subset of polypeptides that are proteolytic cleavage victims in the cell death pathway. Finally, persistent viral infections may be sustained and propagated because the normal host cell suicide response that would be engaged following viral infection is cleverly suppressed by antiapoptotic viral gene products. Nicholson, supra.
Many of the clues which have implicated various components of the cell death pathway have arisen from disease association, genetic analysis and in vitro reconstitution of apoptotic events. Collectively, this information has helped define a biochemical pathway that accounts for many of the key events that occur in dying cells in vivo. Many parts of this pathway, particularly those involved in the effector events that mediate the actual cell death process itself, appear to be common to most cell types. At the heart of this process, proteases related to mammalian interleukin-1xcex2 converting enzyme (ICE) and to nematode CED-3 appear to play an essential role. Several of the substrates that are cleaved by ICE/CED-3 like proteases at the onset of apoptosis have also been identified. These include proteins that function in DNA repair as well as structural proteins and regulatory enzymes. A fundamental principle of apoptotic cell death thus appears to be the proteolytic disabling of key homeostatic- and repair-processes as well as the obvious structural dismantling of the cell that is required to facilitate its breakdown and subsequent packaging into apoptotic bodies. Moreover, ICE/CED-3 like proteolytic activities have been demonstrated to play a role in most if not all of these cleavage events. Nicholson, supra.
Molecular cloning has identified several human homologues of ICE and CED-3 (CEll Death gene 3 protein) including ICErel-II (TX, ICH-2), ICErel-III, ICE-1, CPP32 (apopain, Yama) Mch2 and Mch3 (ICE-LAP3) (Munday et at, J. Biol. Chem. 270:15870 (1995); Faucheu et at, EMBO J. 14:1914 (1995); Kamens at al., J. Bio. Chem. 270:15250 (1995); Kumar et al., Biochem. Biophys. Res. Commun. 185:1155 (1992); Kumar at al., Genes and Develop. 8:1613 (1994); Wang et al., Cell 78:739 (1994); Fernandes-Alnemri et al., J. Biol. Chem. 269:30761 (1994); Fernandes-Alnemri et al., Cancer Res. 55:2737 (1995); Fernandes-Alnemri et al., Cancer Res. 55:6045 (1995); Duan at al., ICE-LAP3 for ICE-Like Apoptotic Protease 3, a novel mammalian homolog of the Caenorhabditis elegans cell death protein CBD-3 is activated during Fas- and tumor necrosis factor-induced apoptosis, J. Biol. Chem. in press (1996)). Nicholson, supra.
However, it would be desirable to identify other mammalian (including human) ICE/CED-3 like proteases.
In accordance with the present invention, polynucleotides encoding a novel protease, FMH-1, and other related proteins are disclosed. xe2x80x9cFMH-1xe2x80x9d is used throughout the present specification to refer to both proteins and polynucleotides encoding those proteins and to refer to proteins and polynucleotides from all mammalian species.
In certain embodiments, the present invention provides for an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence of SEQ ID NO: 1 from nucleotide 121 to nucleotide 1683;
(b) a nucleotide sequence capable of hybridizing to a nucleic acid sequence specified in (a);
(c) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 and varying from the sequence of the nucleotide sequence specified in (a) as a result of degeneracy of the genetic code;
(d) a nucleotide sequence comprising a fragment of (a) which encodes an amino acid sequence comprising amino acids 399 to 403 of SEQ ID NO: 2;
(e) a nucleotide sequence comprising a fragment of (a) which encodes an amino acid sequence comprising amino acids 20 to 95 of SEQ ID NO: 2; and
(f) an allelic variant of the nucleotide sequence specified in (a).
In preferred embodiments, the nucleotide sequence encodes for a protein having an activity selected from the group consisting of FMH-1 protease activity and adaptor binding activity. In other embodiments, the nucleotide sequence is operably linked to an expression control sequence. Preferably, the polynucleotide encodes a human FMH-1 protein. Particularly preferred embodiments include polynucleotides comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 121 to nucleotide 1683; a fragment of such sequence which encodes an amino acid sequence comprising amino acids 399 to 403 of SEQ ID NO: 2; and a fragment of such sequence which encodes an amino acid sequence comprising amino acids 20 to 95 of SEQ ID NO: 2.
The invention also provides for a host cell transformed with such polynucleotides, including mammalian cells. A process is also provided for producing an FMH-1 protein, said process comprising: (a) growing a culture of the host cell of the invention in a suitable culture medium; and (b) purifying the FMH-1 protein from the culture. Proteins produced according to such processes are also provided.
Pharmaceutical compositions are also provided comprising such polynucleotides and a pharmaceutically acceptable carrier. In preferred embodiments, the polynucleotide is contained in a vector suitable for gene therapy and/or the composition further comprises agents capable of increasing the uptake of said polynucleotide by cells.
The invention further provides an isolated FMH-1 protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 2;
(b) fragments of (a) having FMH-1 protease activity; and
(c) fragments of (a) having adaptor binding activity.
Preferred embodiments include proteins comprising the amino acid sequence of SEQ ID NO: 2, proteins comprising amino acids 399 to 403 of SEQ ID NO: 2; and proteins comprising amino acids 20 to 95 of SEQ ID NO: 2. Pharmaceutical compositions comprising such proteins and a pharmaceutically acceptable carrier are also provided.
The present invention also provides compositions comprising an antibody which specifically reacts with an FMH-1 protein of the invention.
The invention also provides for methods of treating conditions associated with excessive or insufficient apoptosis by administering to a mammalian subject a therapeutically effective amount of a pharmaceutical composition comprising an FMH-1 protein (as protein or in the form of a polynucleotide, for example, through gene therapy).